The present invention relates to the demonstration of genes which are involved in the molecular pathways of tumor suppression and to the use of the genes which have thus been demonstrated for treating certain genetic malfunctions, in particular cancers.
The present invention was made possible by isolating cDNA which corresponded to the messenger RNAs which are expressed or repressed during the process of apoptosis which is induced by the p53 suppressor gene.
A global analysis of the molecular events which take place during the cell cycle at the time of development and cell apoptosis is required in order to better understand the importance of the p53 gene in the process of tumor suppression or, on the contrary, of canceration.
The transformation of a normal cell into a tumor cell is a process which takes place in several stages and which requires a sequence of molecular events. At the physiological level, these events find expression in the tumor cell becoming independent of external signals and in an internal deregulation which leads to uncontrolled growth.
Two groups of genes are responsible for this so-called xe2x80x9cmalignantxe2x80x9d transformation; on the one hand oncogenes and, on the other hand, suppressor genes or anti-oncogenes. Because of their deregulation in cancer (resulting most frequently from a mutation or a translocation), oncogenes induce a positive signal which promotes neoplastic growth. By contrast, the suppressor genes are unable, either because they have been deleted, because they are not being expressed due to mutation of the promoter, for example, or because of mutations which modify the structure and function of the protein, to supply, in the cancer, the signal which would normally retard this abnormal growth. As a consequence, malfunction of the suppressor genes contributes to neoplastic transformation.
The object of the present invention is to isolate genes which normally play a part in tumor suppression and any possible malfunctions of which can then be monitored and treated.
In particular, isolation of these genes makes it possible to carry out a gene replacement therapy or else to synthesize protein or non-protein pharmacological agents which, directly or indirectly, induce activation and expression of these genes by acting on the promoters, or else to synthesize pharmacological agents which mimic the physiological effect of these suppressor genes.
The final objective is either to inhibit tumor growth or, even better, induce the apoptotic process in these tumor cells, that is to cause the tumor cells to xe2x80x9ccommit suicidexe2x80x9d.
Thus, in one embodiment the invention relates to an isolated DNA molecule comprising a sequence selected from the group consisting of:
(a) a nucleotide sequence of one of SEQ ID Nos. 4 to 11,
(b) a nucleotide sequence that hybridizes with one of the sequences of
(a), and
(c) a nucleotide sequence that is at least 80% homologous with a
sequence of either (a) or (b). Expression of this DNA molecule may activate cell apoptosis and/or tumor suppression.
In another embodiment, the invention relates to an isolated DNA molecule comprising a sequence selected from the group consisting of:
(a) a nucleotide sequence of one of SEQ ID Nos. 1 or 3;
(b) a nucleotide sequence that hybridizes with one of the sequences of
(a), and
(c) a sequence that is at least 80% homologous with a sequence of either
(a) or (b). Tumor suppression induces expression of this DNA molecule.
In another embodiment, the invention relates to an isolated DNA molecule comprising a sequence selected from the group consisting of:
(a) SEQ ID No. 2,
(b) a nucleotide sequence that hybridizes with SEQ ID No. 2, and
(c) a nucleotide sequence that is at least 80% homologous with either (a) or (b); wherein cell apoptosis induces expression of this DNA molecule.
In a preferred embodiment, the DNA molecule of this invention is SEQ ID No. 11 or fragment thereof.
In yet another embodiment, the invention relates to a biologically functional vector comprising one of the above described DNA molecules. Another embodiment relates to a host cell stably transformed with this vector. In yet another embodiment, the invention relates to a protein obtained by culturing the host cell under appropriate nutrient conditions so as to allow the cell to express the protein.
In another embodiment, the invention relates to a pharmaceutical composition comprising the above described vector and a pharmaceutically acceptable carrier. Another embodiment relates to a method of preventing tumorigenesis, the method comprising contacting cells with a tumorigenesis inhibiting amount of the pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition comprises the DNA SEQ ID NO. 11 or TSAP 3.
In other embodiments, the invention relates to a DNA probe or a PCR amplification primer comprising a nucleotide sequence selected from the group consisting of SEQ ID No. 1-11, or a fragment thereof.
The present invention relates to demonstrating genes which are involved in this apoptosis, particularly TSAP 3, which has been discovered to directly activate apoptosis. Thus, each cell contains within itself a program of physiological death. This is also a physiological process which is involved in development for the purpose of maintaining homeostasis of the body and of preventing abnormal cell proliferations from becoming established even if, for all that, they are not malignant in nature.
One of the most important suppressor genes involved in apoptosis is the p53 gene. In its normal function, this gene controls cell growth and the apoptotic process; in particular, it is this gene which blocks cell growth and which is responsible for inducing the apoptotic process in order to avoid the development of a cancer. Thus, it has been demonstrated that mice which are nullizygous for p53 are much more sensitive to the formation of tumors. The fact has also been demonstrated that, in cancers, the p53 gene is very often altered and leads to the production of proteins which are unable to serve as a vehicle for the apoptotic message.
It is this distinctive feature which has been employed within the context of the present invention.
Thus, the present invention is based on the observation that it is not possible, or that it at least appears very difficult, to institute a direct replacement therapy when the p53 gene is malfunctioning. Thus, when p53 is mutated as it is in cancer, it nullifies the physiological effect of the normal p53.
It was therefore necessary, at least initially, to abandon a replacement therapy which acted directly at the level of p53.
The present invention is therefore linked to studying the genes which are situated downstream of p53 in order to bypass the abovementioned difficulty.
In order to isolate the genes which are activated or inhibited by normal p53 (wild-type p53), a global screening was carried out of gene expression in a cell in which apoptosis had been induced and in the same malignant cell, more specifically in a cell which was expressing a p53 whose function was normal and in a cell which was expressing a p53 which was mutated and whose function was oncogenic. Comparison of the expressed genes (messenger RNAs expressed in the two types of cell) made it possible to identify genes which were expressed differentially, that is which were expressed in one of the cells but not the other (the genes can be activated or inhibited).
It was readily deduced that these genes are involved in the process of canceration, in the one case by their absence, and in the other case by their presence.
The method used for carrying out this differential study is the method described in 1992 by Liang and Pardee (Differential display of eucaryotic mRNA by means of a polymerase chain reaction) Science 257: 967-971 (1992), which is herewith incorporated by reference.
Until now, genes involved in suppression have been isolated either by positional cloning or by using double hybrids. The first method has made it possible, by making a statistical computation, to calculate the greatest probability of where a suppressor gene which was a candidate for a rather specific type of cancer, in particular those of familial origin, might be located within the chromosome. The double hybrid system enables the proteins which interact with a given gene to be isolated one by one.
The approach to the problem which was adopted in accordance with the present invention made it possible to isolate sequences which were directly linked to a function. As a result, in contrast to the random sequencing of the ESTs, the sequences are sequences whose function is known and which arc involved in the apoptosis process which is induced by the p53 suppressor gene.
More precisely, this method was used on a cell model described by Moshe Oren; this model involves mouse myeloid tumor cells which have been transfected with a stable mutant of the p53 gene. Expression of this gene is temperature-sensitive, i.e. when the cells are cultured at 37xc2x0 C., the protein which is produced is a mutated protein, that is to say it cannot act as a tumor suppressor and the corresponding cell line therefore develops in the form of malignant cells; by contrast, at a temperature of 32xc2x0 C., the p53 protein which is expressed is able, like the natural protein, to act as a suppressor, and prevents the corresponding cell line from becoming malignant.
This systematic study made it possible to identify the genes which are involved in the suppression cascade which is induced by p53. More particularly, the inventors discovered that one gene, TSAP3, is responsible for apoptosis and/or tumor suppression.
For this reason, the present invention relates to these novel sequences and the genes which comprise them, as well as to the use of these sequences, both in diagnosis and therapy, and also for creating models for testing antineoplastic products.
The present invention relates, first of all, to a nucleotide sequence which corresponds to a gene which comprises:
(a) a sequence according to one of the SEQ ID Nos 1 to 10, or an equivalent gene which comprises:
(b) a sequence which hybridizes with one of the sequences according to (a),
(c) a sequence which exhibits at least 80% homology with (a) or (b), or
(d) a sequence which encodes a protein which is encoded by a gene according to (a), (b) or (c), or which encodes an equivalent protein,
and their application, in particular in the suppression of cancer and in the therapeutic follow-up.
In addition, the present invention relates to a human gene which is involved in the suppression cascade induced by p53, and to the use of the sequences of this gene, both in diagnosis and in therapy, and also for creating models for testing antineoplastic products and their application as antiviral agents.
The present invention therefore also relates to a nucleotide sequence which corresponds to a gene which comprises:
(a) a sequence according to SEQ ID Nos 11, corresponding to the human TSAP 3 gene or HUMSIAH (Human Homologue of the Drosophila seven in absentia gene), or an equivalent gene which comprises:
(b) a sequence which hybridizes with one of the sequences according to (a),
(c) a sequence which exhibits at least 80% homology with (a) or (b), or
(d) a sequence which encodes a protein which is encoded by a gene according to (a), (b) or (c), or which encodes an equivalent protein,
and their application, in particular in the suppression of cancer and in the therapeutic follow-up.
With regard to sequences 1 to 11, the present invention covers both the nucleotide sequence which corresponds to the entire gene and fragments of this gene, in particular when they encode an equivalent protein, as will be described below.
The nucleotide sequences can equally well be DNA sequences or RNA sequences or sequences in which some of the nucleotides are unnatural nucleotides, either in order to improve their pharmacological properties or to enable them to be identified.
The sequences mentioned in (b) (for SEQ ID Nos 1 to 11) are essentially sequences which are totally or partially complementary (in particular in the previously mentioned cases).
The (a) and (b) sequences (for SEQ ID Nos 1 to 10) provide access not only to the murine gene from which they are derived but also, by homology, to the corresponding human genes.
Thus, the invention also relates to the nucleotide sequences of the genes which exhibit strong homology with the previously mentioned genes, preferably a homology which is greater than 80% over the essential parts of the said genes, or, in general, at least 50% of the sequence; preferably, the homology over these parts is greater than 90%. xe2x80x9cHomologyxe2x80x9d means the degree to which the sequences contain the same nucleotides when two nucleotide sequences are aligned and compared, using methods well known in the art of the invention.
Finally, when the said genes encode a protein, the present invention also relates to the sequences which encode the same protein, taking into account the degeneracy of the genetic code, and also equivalent proteins, that is to say which produce the same effects, in particular proteins which have been deleted and/or which have undergone point mutations.
The sequences according to the present invention are, more specifically, the sequences which are induced or inhibited at the time of cell apoptosis, in particular those which are induced by p53, or which are responsible for apoptosis, as in the case of TSAS 3.
The said genes are grouped together in the TSAP or xe2x80x9cTumor Suppressor Activated Pathwayxe2x80x9d and designated TSAP 1 to TSAP 8 and human TSAP 3, corresponding to SEQ ID Nos 1 to 8 and 11 (HUMSIAH) respectively, and the TSIP or xe2x80x9cTumor Suppressor inhibited Pathwayxe2x80x9d and designated TSIP 1 and TSIP 2, corresponding to SEQ ID Nos 9 and 10.
The characteristics of the sequences which correspond to SEQ ID Nos 1 to 10 are compiled in the appended table.
The nucleotide sequences which correspond to the TSAP genes (including human TSAP 3 or HUMSIAH) are sequences which are expressed during the apoptosis process, whereas the process of oncogenesis takes place when they are not expressed. It is therefore of interest:
to detect any anomaly in the corresponding gene which might lead to greater susceptibility to oncogenesis, and
to be able to plan a replacement therapy.
It must also be recalled that these genes are able to intervene in other processes besides oncogenic processes; thus, p53 is, as it were, the guardian of the integrity of the genome; under these conditions, the TSAP or TSIP genes are doubtless also involved in this control function; the previously mentioned detection and therapy can therefore cover all the possible alterations of the genome. By contrast, the TSIP genes are expressed during oncogenesis and not during apoptosis; it is therefore also of interest in this case to detect any possible anomalies in the TSIP genes and to plan an inhibition/blocking therapy.
The replacement therapy can be effected by means of gene therapy, that is by introducing the TSAP gene together with the elements which enable it to be expressed in vivo. The principles of gene therapy are known. Specific viral or nonviral vectors can be used, for example adenovirus, retrovirus, herpesvirus or poxvirus vectors. Most of the time, these vectors are used in defective forms which serve as TSAP-expressing vehicles, with or without integration. The vectors can also be synthetic vectors, that is to say which mimic viral sequences, or else consist of naked DNA or RNA in accordance with the technique developed by the VICAL company, in particular.
In most cases, it is necessary to provide targeting elements which ensure expression which is specific for tissues or organs; thus, it is not possible to consider activating a phenomenon of uncontrolled apoptosis.
The present invention therefore relates to all the previously described vectors.
The present invention also relates to the cells which are transformed by an expression vector such as previously described as well as to the protein which can be obtained by culturing transformed cells.
The expression systems for producing proteins can be either eucaryotic systems, such as the preceding vectors, or procaryotic systems in bacterial cells.
One of the important features of the present invention is that it has demonstrated the involvement of several genes in apoptosis; thus, the use of gene therapy to over-express one of the genes may, for some of the genes, only lead to apoptosis of the cells in which other deregulated genes are already being expressed, that is malignant cells.
The present invention also relates to a compound, as a medicament, which ensures cellular expression of at least one of the previously mentioned nucleotide sequences, in particular of the TSAP 1 to TSAP 8 and human TSAP 3 genes, when it is induced during cell apoptosis, or, on the contrary, which ensures inhibition of the cellular expression of at least one cell sequence such as previously described, in particular TSIP 1 and TSIP 2, when it is inhibited during cell apoptosis.
It is, for example, possible to envisage approaches other than gene therapy, in particular the use of nucleotide sequences in a sense or antisense strategy, that is to say sequences which are able to block TSIP expression or which, on the contrary, acting upstream, promote TSAP expression.
It is also possible to envisage a direct replacement strategy which involves supplying proteins which correspond to TSAP or inhibitory antibodies which correspond to TSIP.
Finally, it is possible to envisage using non-protein molecules whose activity is to activate TSAP or to mimic the action of its expression product or else to inhibit TSIP or else to block the action of its expression product.
These products can be easily tested on modified cells, which are described in the examples, by introducing the products to be tested into the cell culture and detecting the appearance of the apoptotic phenomenon. In the strategies using DNA, RNA or protein, the products are, of course, developed in accordance with the sequences which are described.
The present invention relates, in particular, to the use of the abovementioned medicaments as antineoplastic agents.
However, the product of the human TSAP 3 gene (HUMSIAH) may also be used as an antiviral agent, as will be apparent from reading Example 2. The present invention therefore also relates to the use of the abovementioned medicaments as antiviral agents.
The present invention also relates to all or part of the sequences according to the invention for use, in the role of a diagnostic agent for determining predisposition to cancer, as a nucleotide probe or as an amplification primer, and, also in the role of diagnostic agent for determining predisposition to cancer, to an antigen which corresponds to all or part of the proteins encoded by the sequence according to the invention or to the corresponding antibodies, in particular monoclonal antibodies, where appropriate following culture.
The diagnostic methods are known; they can, for example, be techniques for microsequencing variable parts following isolation and possible amplification, or detection methods of the RFLP type, or straightforward amplification in particular. The differential techniques can, in particular, make it possible to demonstrate the divergence between normal and abnormal TSAP or TSIP.
The invention also relates to models which make use of the abovementioned sequences. The PCR method, or other amplification methods, may be employed, in particular, to isolate the human TSAP 3 gene (HUMSIAH) by utilizing the structure of the gene. It is also possible to synthesize this gene bit by bit, if required.
Finally, the invention relates to an improvement to the method of Liang and Pardee, Science 257: 967-971 (1992), which involves carrying out a stepwise decrease (xe2x80x9ctouch downxe2x80x9d), as described in Don et al. Nucl. Acids Res. 19: 4008 (1991), in the PCR amplification. Liang et al and Don Et al are herewith incorporated by reference.